Retinal pigment epithelial cell cultures on amniotic membrane and transplantation

ABSTRACT

The present invention relates to a composition for implantation in the subretinal space of an eye, the composition including amniotic membrane, which may be cryopreserved human amniotic membrane, and a plurality of retinal pigment epithelial (RPE) cells or RPE equivalent cells present at the amniotic membrane. The amniotic membrane may be intact, epithelially denuded, or otherwise treated. The invention includes the use of amniotic membrane for the culturing of RPE cells thereon, forming a surgical graft for replacement of Bruch&#39;s membrane as a substrate, and for the transplanting of RPE cells to the subretinal space. The composition does not elicit immunological reactions to alloantigens or to RPE specific autoantigens; and exerts anti-inflammatory, and angiogentic, and anti-scarring effects. The invention includes methods and kits for making or using composites including amniotic membrane and RPE cells. Also disclosed is a device for harvesting RPE cells.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/415,986 filed on 4 Oct. 2002, the teachings of which are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

The retina is a multi-layered nervous tissue where light energy isconverted into nerve impulses. The outermost layer of the retina,closest to the front of the eye, is a layer of neurons that includesganglion cells. Behind the ganglion cells is a layer of integratingneurons, and behind the integrating neurons is a layer of photoreceptorcells, called rods and cones. Photoreception in rods and cones beginswith absorption of light by a pigment in the cells, the absorbed lightcausing a receptor potential.

Forming an intimate structural and functional relationship with thephotoreceptor cells is the retinal pigment epithelium, a monolayer ofspecialized, cuboidal cells located immediately behind the retina. Theretinal pigment epithelial (RPE) cells provide support for thephotoreceptor cells and carry on important physiological functions,including solute transport, phagocytosis and digestion of discardedouter segments of membranes shed from photoreceptor cells, and drugdetoxication.

The RPE cells rest on a specialized basement membrane, called theBruch's membrane, a membrane 1 to 5 microns in thickness and composed ofcollagen, laminin and other molecules.

Underlying the RPE cells is the choriocapillaris of the choroid tissue.The choriocapillaris contains the vasculature to provide nutrients andremove metabolic by-products from the retina. Underlying the choroidtissue is the sclera.

It is believed that failure of the RPE cells to properly perform theirfunctions alters the extracellular environment for photoreceptor cells,and leads to the eventual degeneration and loss of photoreceptor cells.Dysfunction of RPE contributes to the pathogenesis of a variety ofsight-threatening diseases including age-related macular degeneration(ARMD) 1, serious retinal detachment 2, and such genetic diseases asgyrate atrophy 3 and choroideremia 4.

Age-Related Macular Degeneration

ARMD is the leading cause of visual impairment in western countries andis believed to be caused by progressive deterioration of RPE, Bruch'smembrane, and the choriocapillaris, which leads to subsequent damage tothe photoreceptor cells. In ARMD, the RPE cells are dysfunctional. Inone form of ARMD, degeneration of the RPE cells is followed by atrophyof the choriocapillaris. In another form, the Bruch's membrane isaltered and degraded by invasion of choroid neovascular membrane (CNV)into the subretinal space, leading to hemorrhage in the subretinalspace, and scarring, with possible further damage to both RPE andphotoreceptors.

Although CNV invasion 5 into the subepithelial and/or subretinal spacecan be treated with laser photocoagulation, if neovascularization issubfoveal, the results are poor 6. Surgical removal of CNV membranesseldom leads to improvement of vision or halts the progression of ARMD7. The poor results may be due to inadvertent removal of RPE during theCNV removal 8, the failure of RPE to re-populate, the progressiveenlargement of choriocapillaris atrophy following submacular surgery 9,and photoreceptor loss.

Problems with Prior Art Methods of RPE Transplantation

Only limited success in restoring vision using current methods of RPEtransplantation has been achieved with either autologous or allogeneicsources (experimental 6;10 and clinical 11-14). In experimental animals,in particular in the Royal College of Surgeons ARCS) Rat model ofretinal degeneration 15-19, RPE transplantation has been used to rescuephotoreceptors, preserve choriocapillaris, and prevent CNV. In the caseof allogeneic RPE transplantation, one obvious reason to explain thefailure is allograft rejection 13;20. However, in the case of autologousRPE transplantation, the failure to restore vision may be due to thefailure of transplanted RPE to repopulate the diseased site or tofunction in vivo. The failure of the RPE to grow or to function may bedue to damage to the Bruch's membrane.

The Bruch's Membrane

There is evidence that the integrity of Bruch's membrane is crucial forRPE repopulation and subsequent functions. For example, surgical removalof RPE without damage to Bruch's membrane results in partialregeneration of the RPE monolayer in the non-human primate and domesticpig with the preservation of the underlying choriocapillaris and theoverlying photoreceptors 21-23. In contrast, abrasive debridement causesmore damage to Bruch's membrane, leads to incomplete repopulation ofRPE, choriocapillaris atrophy, and outer segment retinal degeneration24. Experimental transplantation of cultured human RPE to Bruch'smembrane of the owl's monkey eye results in normal attachment,viability, and expressing junctions and morphological polarity 25.Autologous transplantation of RPE onto an abrasively debrided Bruch'smembrane decreases choriocapillaris atrophy and photoreceptor loss inrabbits 26.

In the case of human patients with ARMD, the failure of restoring RPEfunction in transplanted human autologous RPE may be at least partiallydue to the altering of Bruch's membrane intrinsically caused by ARMD 27and damaged by surgical removal of CNV membrane 23;24.

The current method of RPE transplantation, subretinal injection of anRPE cell suspension, achieves a limited success. There are many problemsassociated with this method, including a resulting subretinal fibrosisand the formation of multiple layers of RPE 6. These problems may be dueto lack of restoration of in vivo (normal) epithelial phenotype andfunction. To date, no advance has been made in restoring Bruch'smembrane in the surgical treatment of ARMD.

Immunological Aspects of RPE Transplantation

Although the eye as a part of the central nervous system hascharacteristics of an immunologically privileged site, it has beendemonstrated that RPE transplants sensitized their recipients to bothalloantigens and to RPE-specific autoantigens. Both are consideredpotential barriers to successful transplantation, and would make immunesuppression regimens necessary 28. It was also demonstrated that theimmunological response is most likely related to the amount oftransplanted cells and that the response increases with time. RPEallografts in the RCS rat were not rejected for up to one year.

Problems with Prior Art Substrates and Methods of Culturing RPE

Substrates that have been used for this purpose include plastic 31,cross-linked collagen 32, gelatin 1, fibrinogen 2, poly-L-lactic acidELLA) 3, PLLA/PLGA (poly-DL-lactic-co-glycolic acid) film 5-6, hydrogel7, and basement membrane-containing anterior lens capsule 7. There aremany disadvantages associated with each of the prior art substrates usedfor culturing RPE cells for transplantation, and a number of problemsremain unsolved.

Impermeable Substance

One attempt at RPE transplantation utilized RPE cells isolated fromeither the whole eye or from a biopsy with Dispase® (GodoShusei Co.,Ltd., Tokyo, Japan), and seeded on an impermeable substrate such as theplastic dish 31. These cells were prepared as a dissociated cellsuspension 6;10 or as a patch derived from fetus 11 beforetransplantation. These cells did not fully retain their epithelialmorphology. Furthermore, pigmentation of melanolipofuscin granulesrapidly disappears on plastic cultures 33.

Porous Support (Cross-Linked Collagen, Collagen, Gelatin, Fibrinogen,PLLA/PLGA, Hydrogels, CNV Membranes, Lens Capsule)

Cross-linked collagen, when used for transplantation, is damaging to theretina due to its thickness, poor permeability and inability to degrade32. Although human RPE cells 34 seeded on collagen membrane produced amonolayer of cells that exhibited a measurable transepithelialresistance and electrical potential 35, the cells did not achieve the invivo state of development and function.

Gelatin has been used as an embedding medium, but not as a substrate forattachment. Fibrinogen and PLLA microspheres are also not suitable fortransplanting RPE as a single sheet when transplanted to the subretinalspace 2;3. PLLA/PLGA films do provide the RPE monolayer sheet fortransplantation, but in vitro cultures of human fetal RPE cells grown onthese supports do not show pigmentation (melanogenesis) 5;6. Hydrogelalso provides the RPE monolayer sheet for transplantation, but theresultant cell density and the cell tight junction determined byexpression of ZO-1, is relatively low 7.

Human RPE cells have also been cultured on surgically excised CNVmembranes from ARMD patients, but the culture forms multiple layers 36.

Although lens capsule is a basement membrane-containing, naturalmaterial, it is not an ideal substrate for RPE culture andtransplantation. Anterior lens capsule has been used to grow RPE 7;8 andIPE 8, and to transplant RPE and IPE 8 with lens capsule to thesubretinal space. Both hydrogel and lens capsule, when used assubstrates for RPE cultures, do not allow pigmentation to form (ormelanogenesis) by RPE cells in culture 7;8.

The inventors of the present subject matter attempted to use the lenscapsule as an autologous substrate for RPE/IPE transplants. However, thetendency of the capsule to curl made this technique impracticable. Theidea to use the posterior capsule, because it is thinner, was alsoabandoned, for a number of reasons. First, the posterior capsule isdifficult to obtain during surgery, without putting the patient at ahigh risk. Secondly, no absorption or slow absorption of the lenscapsule material might inhibit the survival of the transplanted RPEcells because of insufficient contact of the cells with the Bruch'smembrane and/or choriocapillaris.

Recently Bilbao et al, 37 disclosed the use of PLGA, coated on one sideof a lens capsule to prevent curling and to facilitate its use forsubretinal release. However, histological studies showed not only thatthe PLGA had completely dissolved after 4 weeks, but also that theoverlying retinal layers were disrupted, the disruption accompanied by alarge amount of cell infiltration.

Cryoprecipitate from blood donors was also tested as a possibleautologous substrate for human fetal retinal pigment epithelium byFarrokh-Siar et al in 1999 (38). Dutt et al, 39 used several substratesfor culture of BPE cell line 0041: extracts from placenta and amnion;MATRIGEL® (Collaborative Biomedical Products. Inc., Bedford, Mass.), acommercially available basement membrane matrix; dishes coated withextracellular matrix secreted by endothelial cells (ECM); dishes coatedwith collagen IV and/or laminin; dishes coated with collagen I and/orfibronectin. Although deeply pigmented, cells grown on MATRIGEL® lookedlike fibroblasts.

As described above, problems that remain to be solved include, forexample, maintenance of the morphology of the RPE phenotype in culturedand transplanted RPE cells; creation of a uniform monolayer ofautologous RPE on a biocompatible substrate; improvement of thetransplant technique to better cover the defect; overcoming immunerejection of RPE transplants due to both alloantigens and RPE-specificauto-antigens; and prevention of subretinal fibrosis following RPEtransplantation.

Amniotic membrane is a biological membrane that lines the inner surfaceof the amniotic cavity and comprises a simple, cuboidal epithelium, athick basement membrane, and an avascular mesenchymal layer containinghyaluronic acid. Amniotic membrane transplantation has been used forocular surface reconstruction in the treatment of acute chemical andthermal burns of corneal tissue 53.

Overall, the medical need for a method of culturing RPE cells suitablefor transplantation to the subretinal space, a suitable RPE transplantcomposite with actions to maintain the epithelial phenotype and exertanti-inflammatory, anti-scarring, and anti-angiogenic effects to theunderlying stroma, and a method of transplanting RPE cells to thesubretinal space, has not been met.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to the discovery thatcryopreserved amniotic membrane, when appropriately procured andprocessed, can be used for the culturing of RPE cells or RPE equivalentcells thereon, and as a surgical graft for replacement of Bruch'smembrane as a substrate, and for the transplanting of RPE cells or RPEequivalent cells to the subretinal space; and that the graft does notelicit immunological reactions. The invention relates, in one aspect, tocompositions for implantation in the subretinal space of an eye of apatient in need thereof, the composition including amniotic membrane andRPE cells or RPE equivalent cells. In one embodiment of the invention,the amniotic membrane present in the composite is human amnioticmembrane. The amniotic membrane may be intact, epithelially denuded, orotherwise treated. In one embodiment of the invention, the membrane istreated on one side, for example to thin or remove one side. In anotherembodiment, the amniotic membrane is reshaped by laser ablation toremove the stromal side or to thin the basement membrane side. In yetanother embodiment mesenchymal cells are added to the stromal side. Theinvention, inter alia, comprises the following, alone or in combination.

One embodiment of the invention includes a composite comprising amnioticmembrane; and a plurality of retinal pigment epithelial cells or retinalpigment epithelial equivalent cells present at the amniotic membrane.

Another embodiment includes a kit comprising amniotic membrane; aplurality of retinal pigment epithelial cells or retinal pigmentepithelial equivalent cells present at the amniotic membrane; a buffermedium or a culture medium; and optionally, instructions forsimultaneous, separate, or sequential use of at least one component ofthe kit for treating a retinal disease. In one embodiment, the amnioticmembrane included in the kit is human amniotic membrane.

Another embodiment of the invention is a method of forming a composite,the method comprising the steps of applying at least one retinal pigmentepithelial cell or retinal pigment epithelial equivalent cell to anamniotic membrane; and culturing the retinal pigment epithelial cell orretinal pigment epithelial equivalent cell on the membrane underconditions suitable for growth for a period of time sufficient toproduce a plurality of cultured cells. The amniotic membrane used in oneembodiment may be human. In one embodiment, the composite comprisingcultured RPE cells or cultured RPE equivalent cells and amnioticmembrane is used for implantation in the subretinal space of an eye of ahost in need thereof. The host may be any mammal, for example, a human.

In yet another aspect, an embodiment of the invention includes a methodof inducing an excised or cultured retinal pigment epithelial cell orretinal pigment epithelial equivalent cell to express or to maintain thephenotype of retinal pigment epithelial cells, the method comprising thesteps of contacting amniotic membrane with the retinal pigmentepithelial cell or retinal pigment epithelial equivalent cell; culturingthe retinal pigment epithelial cell or retinal pigment epithelialequivalent cell on the membrane under conditions suitable for growth fora period of time sufficient to produce a plurality of cultured cells;and either contacting the cultured cells with an effective amount of anagent that raises the intracellular calcium ion concentration to a levelsufficient to induce or maintain the phenotype of retinal pigmentepithelial cells; or exposing the membrane comprising the cultured cellsto an air-fluid interface for a period of time sufficient to induce ormaintain the phenotype of retinal pigment epithelial cells. The amnioticmembrane used in an embodiment may be human.

In one embodiment, both the step of contacting the cultured cells withan agent that increases the intracellular calcium ion concentration, andthe step of exposing the membrane including the cultured cells to anair-fluid interface to induce or maintain the phenotype of retinalpigment epithelial cells, are performed sequentially or essentiallysimultaneously. However, in another embodiment, the medium can compriseeither a normal or a high Ca²⁺ concentration with growth factors added.

The present invention also relates, in yet another aspect, to the use ofamniotic membrane to promote the growth and the differentiation of atleast one retinal pigment epithelial equivalent cell to a plurality ofcells that express the phenotype of retinal pigment epithelial cells. Ina particular embodiment, the amniotic membrane is human.

Another embodiment of the invention is a method of delivering aplurality of retinal pigment epithelial cells to a target site in asubretinal space in an individual in need thereof, including the stepsof forming at least one hole in a retina of the individual, or at leastpartially detaching the retina to access the subretinal space; insertingthrough the hole a composite including amniotic membrane and the retinalpigment epithelial cells present at the membrane; and positioning thecomposite at the target site.

The invention also relates to a method for treating a retinal disease,the method including inserting in a subretinal space of a patient inneed thereof a composite comprising amniotic membrane, for example,human amniotic membrane, and a plurality of retinal pigment epithelialcells present at the membrane. The RPE cells may be cultured on themembrane according to a method of the invention. Non-limiting examplesof retinal diseases that are treated according to an embodiment of theinvention are age-related macular degeneration, retinal degeneration,gyrate atrophy, and choroideremia.

Another aspect of the invention is the use of amniotic membrane, whichin one embodiment is human in origin, and at least one retinal pigmentepithelial cell, for the manufacture of a composition for treatment of aretinal disease in a patient suffering from, or at risk of developingthe disease.

The invention also relates to the use of human amniotic membrane topromote growth and differentiation of at least one retinal pigmentepithelial equivalent cell to a plurality of cells that express thephenotype of retinal pigment epithelial cells.

Yet another aspect of the invention is the use of amniotic membrane fortransplanting retinal pigment epithelial cells or iris pigmentepithelial cells to a subretinal space, to prevent or decrease asensitizing of a recipient to alloantigens and to retinal pigmentepithelial-specific autoantigens. In a particular embodiment, theamniotic membrane used in transplanting RPE cells or IPE cells to asubretinal space, to prevent or decrease a sensitizing of a recipient toalloantigens and to retinal pigment epithelial-specific autoantigens ishuman in origin.

The invention also relates to the use of amniotic membrane, includinghuman amniotic membrane, to inhibit fibrosis following transplantationof RPE cells or iris pigment epithelial (IPE) cells to the subretinalspace. Another embodiment of the invention is the use of human amnioticmembrane and at least one RPE cell for the manufacture of a compositionfor treatment of a retinal disease in a patient suffering from, or atrisk of developing the disease.

Many advantages are obtained by the use of amniotic membrane, processedand cryopreserved according to an embodiment of the invention, as asubstrate for the culturing of RPE or RPE equivalent cells thereon. Forexample, a composite comprising the amniotic membrane and RPE or RPEequivalent cells can be used as a surgical graft for substratereplacement and the transplanting of RPE cells in the subretinal space.The composite exerts anti-inflammatory, anti-angiogenic,anti-fiberoptic, and anti-scarring effects, and does not elicitimmunological reactions to either alloantigens or RPE-specificauto-antigens. Further, amniotic membrane used according to anembodiment of the invention promotes growth and differentiation of RPEcells in culture, and maintenance of the morphological appearance of theRPE cells both in culture and following transplantation to thesubretinal space. The composite and method of delivery of RPE cells tothe subretinal space result in a uniform monolayer of RPE cells on abiocompatible substrate having a basement membrane. A compositeaccording to an embodiment of the invention can be used as a surgicalgraft suitable for replacement of Bruch's membrane as a substitute andfor the transplanting of RPE cells to the subretinal space. Embodimentsof the invention provide an improved transplant composite and techniqueto better cover the defect; to overcome immune rejection of RPEtransplants due to both alloantigens and RPE-specific auto-antigens; toprevent sub-retinal fibrosis following RPE transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of illustrative embodiments of the invention, as illustratedin the accompanying drawings.

FIG. 1 is a side elevation view of RPE harvesting cannula 10.

FIG. 2 is an enlarged detailed view 20 of distal 12 of cannula 10.

FIG. 3 is a view of FIG. 2 taken at view 3-3.

FIG. 4 is an end view of tip 12 of cannula 10 taken at view 4-4 of FIG.2.

FIG. 5 is a side elevation view of another embodiment of an RPEharvesting cannula 50 with auxiliary infusion line 52.

FIG. 6 is an enlarged detailed view 60 of distal of 58 of cannula 50.

FIG. 7 is a bottom view 70 of distal of 58 of cannula 50 taken at view7-7 of FIG. 6.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

A description of illustrative embodiments of the invention follows. Itwill be understood that the particular embodiments of the invention areshown by way of illustration and not as limitations of the invention. Atthe outset, the invention is described in its broadest overall aspects,with a more detailed description following. The features and otherdetails of the compositions and methods of the invention will be furtherpointed out in the claims.

The inventors of the subject matter of the present invention attemptedfirst to use the stromal side of amniotic membrane as a substrate onwhich to grow human umbilical vascular endothelial cells, but the cellswould not grow on amniotic membrane. In fact, the endothelial cellsunderwent apoptosis. The inventors also used the stromal side ofamniotic membrane as a substrate in an attempt to grow humanpolymorphonuclear leucocytes thereon, but the leucocytes would not growon amniotic membrane.

The invention relates to the discovery that, under suitable conditions,RPE cells, RPE equivalent cells, and IPE cells will grow onappropriately procured and processed, cryopreserved amniotic membrane,for example, human amniotic membrane. Histologically, amniotic membranecomprises a thick basement membrane and an avascular stroma. Theinventors have discovered that human amniotic membrane is an idealextracellular matrix substrate to promote growth and differentiation ofRPE equivalent cells and RPE cells in culture.

The RPE cells, RPE equivalent cells, and IPE cells grown on amnioticmembrane under suitable conditions, differentiate, tend to retain theirmorphological characteristics, and do not tend to de-differentiate.Further, the amniotic membrane with the cells present thereon can beused as a surgical graft to transplant RPE cells to the subretinalspace, and for substrate replacement. The graft does not elicitimmunological reactions, and can be used to treat retinal diseases.

Accordingly, the invention relates to the resulting composite, includingamniotic membrane, and a plurality of RPE, RPE-equivalent cells, or IPEcells, and to a method of use of the composite as a surgical graft totransplant RPE to the subretinal space of a mammalian eye in order totreat retinal diseases such as age-related macular degeneration, retinaldegeneration, gyrate atrophy, and choroideremia.

RPE or RPE-equivalent Cells

The term, “RPE equivalent cells,” as used herein, refers to cells thatare derived from either retina, iris, ciliary body, adult stem cells orembryonic stem cells, and which either retain their normal phenotype orfunction; or which may have less than optimal function; or which havebeen induced in vitro or in vivo to differentiate into RPE cells.

According to an embodiment of the invention, the source of RPE cells canbe from human or other mammals. According to another embodiment of theinvention, the source of RPE cells can be either autologous (from thesame individual as the recipient) or allogeneic (from a differentindividual from the recipient). For the latter, these cells can beeither obtained from adult or fetal, cadaveric or living individuals, ofwhich the latter can be BLA-matched or non-matched.

Further, the source of human RPE equivalent cells can be derived fromretina or iris or ciliary body. The RPE cells, in one embodiment,comprise cells derived from neural retinal cells, for example, rod cellsor cone cells. If derived from iris, the cells are termed iris pigmentepithelial cells (IPE), and their function may be suboptimal. The sourceof human RPE equivalent cells can also be derived from RPE cells thathave been immortalized by viral or non-viral agents but still retainnormal phenotype or function.

The source of human RPE equivalent cells can also be derived from adultstem cells or embryonal stem cells, of which the differentiation intoRPE has been induced in vitro. For the former, such adult stem cells canbe obtained either autologously or allogeneically from various sites ofthe body, for example, from the peripheral blood or bone marrow.

The source of RPE equivalent cells can also be derived from othernon-human species but bioengineered so that they become compatible withhuman cells. For example, a source of the retinal pigment epithelialequivalent cells used in a composite according to the invention mayinclude at least one bioengineered cell induced in vitro todifferentiate into a retinal pigment epithelial cell.

Harvesting of RPE Cells or RPE Equivalent Cells

If the source of RPE or IPE is autologous, the means of harvest will besurgical biopsy from the tissue site of retina or iris. Autologous RPEequivalent cells are derived from adult stem cells, and can be obtainedfrom the site of interest, e.g., peripheral blood or bone marrow. If thesource of RPE or IPE is allogeneic, either from a living individual, orcadaveric, they will be obtained form donated tissues, respectively.Other sources of allogenic RPE equivalent cells are well known to thoseof skill in the art of tissue culture and transplantation.

One method of harvesting or isolating RPE or IPE cells is conventionaland includes non-enzymatic solutions containing EDTA or EGTA orenzymatic digestion using collagenase or DISPASE® solutions. The methodof harvesting or isolating RPE equivalent cells induced from stem cellsis performed in vitro using growth factors and inducible factors.

In yet another aspect of the invention, an embodiment of a device forharvesting RPE cells was conceived and designed. The device, referred toherein as the “Binder RPE harvester cannula”, the “RPE harvestercannula”, or “the cannula”, minimizes damage to the RPE and maximizesharvest yield.

The device has several features and is described as follows. Referringnow to the drawings, FIG. 1 shows an illustrative embodiment of aharvester cannula 10 having a syringe 18 attached by a flexible orbendable tubing 17 to a conventional, tapered LUER female connectorfitting 16. An aspiration line 15 with a distal 12 is connected to thesyringe 18 and passes through the LUER connector 16. In one embodiment,the outer diameter of the harvester cannula 10 is a standard 20 gauge(ga) (0.9 mm) at the sclerotomy site to match current vitrectomyinstruments. Its proximal end may be terminated with a conventional LUERfemale connector fitting, and a short, for example, about 10 cm to about20 cm long, flexible tube 17 connected to a glass syringe 18, forexample, 0.5 cc capacity. The term “flexible” as used herein means thatthe tube described as “flexible” can be bent by a degree sufficient tomanipulate the device during surgery. The flexible tube 17 is preferablymade of non-stick material such as Teflon to prevent the adhesion of RPEcells to the inner wall and thus maximize harvesting.

The cannula distal 12 is bent (for example, approximately a 10 mm radiusof curvature). Approximately the last 5 mm of the distal tip 12, thatwill be placed in contact with the retinal tissues, has a flattenedcrescent-shaped hollowed cross-section with a radius of curvaturematching the surface of Bruch's membrane at the posterior pole. Thatradius is 11 millimeters (mm) approximately. As shown in FIG. 2, anenlarged detailed view of distal of 12 of cannula 10 shows anapproximately 10 degree taper on the end of tip 12 forming lower lip 26and protruding upper lip 24. Therefore, as the cannula is used insurgery, the upper or higher lip protrudes forward and is visible at alltimes through the operation microscope (surgeon's view). FIG. 2 alsoshows directional views for FIGS. 3 and 4. When the cannula is in use,the protruding upper lip 24 is in contact with the retina, which isslightly lifted; and the lower lip 26 is in contact with RPE cells beingcollected.

In one embodiment, the thickness of each lip is approximately 75micrometers; the hollow bore is of about 100 micrometers in height,making the thickness of the tip about 250 micrometers. The width of thecannula's outer surface at its distal end can be approximately 1.5 mm;the width of the hollow bore can be approximately 1.35 mm. In oneembodiment, the upper or higher lip taper is finished with a smooth,polished round edge of approximately 3 micrometers. As the higher lip isslightly tapered, and as the retinal tissue is somewhat elastic orbendable, the retinotomy width needs to be only 1 mm approximately.

In one embodiment, the RPE harvester cannula has a flattened crescentshape with a forward protruding lip that can glide on the surface of theelastic Bruch's membrane supporting the RPE cells. This will maximizeRPE cell harvesting over a width of approximately 1.2 mm per pass. Asthe posterior curvature of the cannula's lip matches that of Bruch'smembrane, it will minimize damage to the membrane itself That is, littleor no cutting, stripping or pitting can occur. Because itscross-sectional shape is similar to the length and shape of theretinotomy opening, a good fit will be obtained between the cannulaouter wall and the edges of the retinotomy. This will minimize trauma tothe retina and prevent or minimize backflush of RPE cells into thevitreous cavity. For example, in FIG. 3, a view of FIG. 2 taken at view3-3 shows upper protruding lip 24, lower lip 26, of a hollow in-tube 30forming aspiration 32.

The RPE harvester cannula can be made of stainless steel and preferablyof anti-stick plastic, such as Teflon-like materials. An illustrativeembodiment utilizes chloro-trifluoroethylene (CTFE) plastic which hasthe advantage of being totally transparent, allowing the surgeon to seethe content of the cannula's inner bore.

Another embodiment of the RPE harvesting cannula 50, shown in FIG. 5,has an aspiration line 56, and an auxiliary infusion line 52 connectedto its upper body. Line 52 has an out-infusion 59 situated at the startof the curvature of the tip of the cannula. An infusion LUER 57 isconnected to a 1 cc syringe 54 via a short flexible or bendable line 17.In use, when the tip of the harvesting cannula is beneath the retina, ashort pulse-like bolus of saline is injected from syringe 54, throughinfusion line 52 , which may be made of stainless steel and soldered tothe aspiration line 56. The bolus of saline lifts the retina and thusforms a bleb, that is, a space or tent under the retina, allowing thesurgeon to harvest RPE cells with greater ease. If not used, theinfusion 59 should be closed by a LUER plug to prevent back-flush.

FIG. 6 shows an enlarged view 60 of distal of 58 of cannula 50 showingthe same profile as FIG. 2 with the addition of infusion line 52 andinfusion 59.

FIG. 7 is a bottom view 70 of distal of 58 of cannula 50 taken at view7-7 of FIG. 6, and showing added infusion line 52 and out-infusion 59.

Method of Preparing Amniotic Membrane

Methods of preparing cryopreserved human amniotic membrane suitable foruse in an embodiment of the invention are well known in the art and aredescribed, for example in U.S. Pat. Nos. 6,152,142 and 6,326,019 B1 toTseng, the teachings of each of which are incorporated herein byreference in their entireties. Methods of preservation of amnioticmembrane are also described in WO 01/08716 A1, the teachings of whichare incorporated herein by reference in their entirety. The amnioticmembrane can also be freeze-dried.

Amniotic membrane suitable for use in an embodiment of the invention isobtained from mammalian placenta, especially human placenta, from whichthe chorion has been separated. The amniotic membrane used in anembodiment may also be derived, for example, from an equine, a bovine,or an alpaca source. Amniotic membrane suitable for use in an embodimentof the invention generally includes an epithelial layer, a basementmembrane, and a stroma, the combination of the three layers preferablyhaving an average total thickness of about 200 μm. Sheets of theamniotic membrane can be cut to size, mounted on filter paper, andstored in a storage solution. Such sheets can also be cut to sizewithout being mounted on filter paper so long as the side of the surfaceis marked. If freeze-dried, the freeze-dried sheet is not stored in asolution. The storage solution comprises a culture medium and ahyperosmotic agent, wherein the hydration of the amniotic membrane ismaintained. The membrane can be impregnated with therapeutic agents,prior to storage or prior to use.

For use in an embodiment of the invention, the amniotic membrane iseither intact (i.e., without additional treatments) or epitheliallydenuded (i.e., by EDTA and mechanical means as reported previously 62).See, Grueterich M, Espana E, Tseng SCG; Connexin 43 expression andproliferation of human limbal epithelium on intact and denuded amnioticmembrane, Invest Ophthalmol Vis. Sci., 43:63-71 (2002), incorporatedherein by reference in its entirety. The amniotic membrane is eitherintact or ablated to remove the stromal portion on the stromal surface.If the epithelially denuded membrane is to be used, the denuded membraneis prepared before being seeded with RPE or RPE equivalent cells inculture (see below). However, if the amniotic membrane stroma is to bethinned, the stroma should be ablated either before or after suchculturing. The method of ablation can be laser-driven, for example, byexcimer laser. In other embodiments, the amniotic membrane can betreated to enable the RPE cells to better adhere to the membrane. Forexample, the membrane can be treated to produce an electrical chargethereon.

Method of Culturing RPE or RPE Equivalent Cells on Amniotic Membrane

After harvesting, the RPE or RPE equivalent cells are cultured in amedium containing culturing supplement including serum and growthfactors. In one embodiment, the medium comprises a low Ca²⁺concentration from about 0.01 millimolar (mM) to about 0.4 mM preferably0.1 mM. In another embodiment, the medium can comprise a normal or ahigh Ca²⁺ concentration with growth factors added. The expansion cultureis performed on a culturing substrate of choice. According to anembodiment of the invention, the expansion culture is performed oncryopreserved amniotic membrane.

A standard culturing method is used according to an embodiment of theinvention. Cell seeding density can be varied depending on the surfacearea of amniotic membrane used. RPE or RPE equivalent cells aregenerally removed from a plastic substrate when they reach thesubconfluent stage by conventional methods utilizing trypsin and/orEDTA. The isolated RPE or RPE equivalent cells are seeded on theamniotic membrane on the epithelial side, with the basement membraneeither exposed or still covered by intact amniotic epithelial cells.

Methods of Inducing Epithelioid Phenotype in Culture of RPE on AmnioticMembrane

According to an embodiment of the method, the step of culturing theretinal pigment epithelial cell or retinal pigment epithelial equivalentcell on the membrane is continued until the cells reach confluence.Ideally, the number of retinal pigment epithelial cells present at themembrane is about 4000 cells per 1 mm². However, the ideal number willdepend on the size of the defect to be covered with the transplantedcomposite. For example, from about 16,000 to about 20,000 cells withhigh vitality are needed to cover a 4 mm² defect.

One method according to an embodiment of the invention, of inducingepithelial phenotype from a fibroblastic phenotype of RPE, is to elevatecalcium concentration from low (for example, a range of from about 0.01to about 0.4 mM, preferably about 0.1 mM) to high in the range of fromabout 0.5 to about 2.0 mM, preferably about 1.8 mM. The calcium ionconcentration may be elevated, according to an embodiment, by adding asoluble calcium salt to the culture medium. Alternatively, an agent suchas a calcium ionophore, which facilitates transport of calcium ionacross the lipid barrier of the cell membrane by combining with the ionor by increasing the permeability of the barrier to the ion may be usedto increase Ca²⁺ concentration. Another embodiment includes an agentthat increases intracellular calcium concentration by blocking theexport of Ca²⁺ out of the cytoplasm. In another embodiment, the mediumcan comprise a normal or a high Ca²⁺ concentration with growth factorsadded. According to another embodiment, the amniotic membrane comprisingRPE or RPE equivalent cells cultured thereon is exposed to air-fluidinterface. A combination of methods can also be employed, eithersimultaneously or sequentially.

The Composite Including RPE Cells or RPE Equivalent Cells on AmnioticMembrane

According to one embodiment, a composite includes intact human amnioticmembrane comprising a basement membrane and a stroma. In anotherembodiment of the invention, the human amniotic membrane of thecomposite is epithelially denuded.

The invention also relates to a composite that further includes at leastone pharmaceutically active molecule. In one embodiment of theinvention, the pharmaceutically active molecule in the composite is oneor more of the following: a growth factor, an enzyme, or a therapeuticdrug.

A Kit for Treating Retinal Disease

Another embodiment includes a kit comprising amniotic membrane, whichmay be human amniotic membrane; a plurality of retinal pigmentepithelial cells or retinal pigment epithelial equivalent cells presentat the amniotic membrane; a buffer medium or a culture medium; andoptionally, instructions for simultaneous, separate, or sequential useof at least one component of the kit for treating a retinal disease. Ina particular embodiment, the kit further includes at least onepharmaceutically active agent. The agent may include growth factors,enzymes, and therapeutic drugs. The growth factor may include retinalpigment epithelium-derived growth factor and/or transforming growthfactor-beta. The agent may include interleukin-10. The agent may bepresent on the composite, or separately packaged, to be added to thecomposite or to the target tissue site in the subretinal space prior toimplantation, or to be administered to the patient subsequent totransplantation.

The kit according to an embodiment may include retinal pigmentepithelial equivalent cells that are bioengineered cells.

The kit according to an embodiment may include a composite formed ofamniotic membrane and retinal pigment epithelial equivalent cells ofautologous origin that have been previously harvested from the intendedrecipient, sent to a laboratory wherein the cells are cultured onamniotic membrane, and added into the composite.

Method of Transplant of RPE Cells to the Subretinal Space

The surgical procedure for transplant of RPE is similar to standardprocedure for intraocular surgery. The procedure comprises the followingsteps:

-   a) Pars plana vitrectomy and removal of the posterior hyaloid    membrane;-   b) The first retinotomy is performed temporally or nasally superior    to the CNV membrane;-   c) Using Ringer's solution, the submacular CNV membrane is gently    hydro-dissected and removed with a sub-retinal forceps. During this    step, the intraocular pressure is elevated, and Perfluorocarbon    (PFCL) is used to prevent or minimize bleeding.-   d) The intraocular pressure is lowered to about 15 to about 20    millimeters (mm)Hg, and a shallow retinal detachment is created, if    not present, by sub-retinal injection with Ringer's solution;-   e) With the help of either an injection system or a specially made    forceps having very smooth surfaces, such as, for example,    mirror-finish or anti-stick Teflon surfaces, the prepared sheet of    amniotic membrane with RPE monolayer is delivered into the    sub-retinal area in the foveal area;-   f) Finally, an air or gas tamponade is made to secure the transplant    sheet in position; the scierotomies are closed; and the patient is    asked to remain in a prone position for the next few days.

With regard to the forceps that can be used in step “e”, it should benoted that unmodified, commercially available forceps cannot be easilyused to transfer the amniotic membrane-RPE implant to a location underthe retina, because the membrane sticks to stainless steel so well thatthe implant cannot be released. Therefore, the jaws should be coatedwith a Teflon®-like substance. Note that the Teflon coated jaws must bemirror-finished as any micro asperities will attach or stick to themembrane, thereby preventing release of the membrane in the spacesurgically created between the retina and Bruch's membrane. A prototypeforceps comprising chlorotrifluoroethylene (CTFE ) instead of stainlesssteel is being made in our laboratory for use in the implantation of theamniotic membrane-RPE composite. CTFE has almost the same anti-stickproperties of Teflon® and is transparent, allowing the surgeon to seethrough it. An additional advantage provided by the CTFE forceps is thatbecause CTFE is more flexible and softer than stainless steel, the CTFEjaws will minimize trauma to the amniotic membrane-RPE implant. Theforceps with flexible transparent CTFE jaws is about 20 gauge (ga).

For the CTFE forceps, we will use one of the many conventionalmechanisms to actuate the jaws. These mechanisms are located in thehandle or immediately in front of the handle which is connected to atube that is about 35 to 50 mm long and has an external diameter ofabout 0.9 mm or less. The function of the tube is to facilitate passingthe forceps through the sclerotomy incision. The tube is made longenough to reach the posterior pole of the eye. The eye is normally about24 mm in length, but depending on the presence of conditions such asmyopia, staphyloma, etc, may be 20 to 35 mm in length. When closed, thejaws have a diameter equal to or a little less than the outer diameterof the tube.

Compressing the handle either retracts the jaws into the tube, therebyclosing the jaws; or moves the tube forward, thereby closing the jaws.If the tube is moved forward, the tip of the jaws remains at a constantdistance from the tissues or object being held. In some handles, arotating knob located either on the distal end of the handle orimmediately in front of it allows for rotation of the jaws. Otherhandles are round and can be easily turned around using one hand. Assurgeons have individual preferences for handles, we will manufactureboth types. All handles are commercially available (e.g., Storz-B&L Inc,Katena Inc, DORC Inc, Grieshaber-Alcon Inc, etc), but the CTFE jaws arenot.

Alternatively, in step “e”, an injection system can be used in lieu ofthe use of forceps to deliver the prepared sheet of amniotic membranewith RPE cells to the sub-retinal area.

It should be noted that, for cell harvesting only, a second retinotomyon the nasal side of the retina is performed.

EXEMPLIFICATION

Animals

Dutch belted rabbits were obtained from Covance Research Products, Inc.(Denver, Pa., USA). All rabbits used were euthanised by intramuscularinjection of 0.3 ml ketamine (35 mg/kg) and xylazine (5 mg/kg) followedby an injection of 1 ml of Euthasol® (Delmarva Laboratories, Inc.,Midlothian, Va.).

Materials

Dulbecco's modified Eagle's medium with F12 nutrient mixture 1:1 (V/V),dialyzed fetal bovine serum (FBS) with molecular cut off rate of 10,000daltons, L-glutamine, L-methionine, L-lysine, L-leucine, magnesiumchloride, magnesium sulfate, calcium chloride, cell culture grade water,sodium bicarbonate, FITC-conjugated goat anti rabbit IgG, and mousemonoclonal anti-cytokeratin (CK) 18 (clone CY-90) were all obtained fromSigma-Aldrich Chemical Company (St. Louis, Mo., USA). Phenol red sodium,DMEM/F12, sterile phosphate buffered saline (PBS), amphotericin B,trypan blue stain solution, and trypsin/EDTA were purchased from GibcoBRL (Grand Island, N.Y., USA). Twenty-four well plates were used.(Corning Life Sciences) Collagenase type 1 with 239 U/mg was purchasedfrom Worthington Biochemical Corporation (Lakewood, N.J., USA).Penicillin and streptomycin were obtained from Bio Whittaker(Walkersville, Md., USA). Goat anti-rat Alexa 546-conjugated IgG (H+L),F(ab)2, Goat anti-mouse Alexa 488-conjugated IgG F(ab)2 were purchasedfrom Molecular Probes Inc. (Eugene, Oreg., USA). Aqua-Poly/mount, wasobtained from Polysciences Inc. (Warrington, Pa., USA). The polyclonalrabbit anti-RPE-65 antibody was a generous gift from T. Michael Redmond.The monoclonal rat anti-ZO-1 antibody (MAB 1520) was obtained fromChemicon (Temecula, Calif., USA). The monoclonal mouseanti-Pancytokeratin K8.13 Ab was obtained from ICN Biomedicals, Inc.(Aurora, Ohio, USA). As previously published 13, for the epithelialdenudement of the amniotic membrane a corneal epithelial scrubber wasemployed (Amoils Epithelial Scrubber; Innova, Innovative ExcimerSolutions, Inc., Toronto, Ontario, Canada). Culture plate inserts usedfor fastening amniotic membrane were from Millipore (Bedford, Mass.,USA).

Primary RPE Cultures

Following euthanasia, eyes were immediately enucleated, and the anteriorsegment was removed by circumferential incision with scissors ca. 3-4 mmposterior to the corneal limbus. The isolation of RPE followed what hasbeen reported 48 except that collagenase was used. In brief, the neuralretina was detached by subretinal injection of sterile PBS, pH 7.4, tofacilitate the removal of the vitreous residues and the neural retina.The RPE surface was then rinsed 3 times with PBS, and the eyecup wasincubated with 1 mg/ml collagenase type 1 in DMEM/F12 for 1 h at 37° C.in the incubator with 5% CO2. RPE sheets were collected by gentleshaving of the Bruch's membrane with a heat-polished glass pipette. Thecells were centrifuged at 800 rpm for 5 min and plated into 24 wellplates (5-6 wells per eye) in DMEM/F12 adjusted to 0.1 mM Ca²⁺,supplemented with 10% dialyzed FBS, 100 IU/ml Penicilin, (100 g/mlstreptomycin and 0.5 g/ml amphotericin B). The cultures were cleared ofthe debris 48 h after plating by changing 50% of the medium with a freshmedium. The medium was then completely changed biweekly.

Amniotic Membrane Preparation

Human amniotic membrane (AM was kindly provided by Bio-Tissue, Inc.(Miami, Fla.) according to the method previously described (U.S. Pat.Nos. 6,152,142 and 6,326,019), and stored in DMEM and glycerol (1:1) at−80° C. before use. Upon use, AM was thawed at room temperature andrinsed with sterile Hanks Balanced Salt Solution (HBSS) to remove excessglycerol, and sutured with 4-0 silk surgical sutures (Alcon Surgical,USA) and/or tightened by a rubber-ring with the epithelial side facingup to 24 well plate culture insert as previously described 63. In aseparate experiment, AM was epithelially denuded by incubation withsterile 0.02% EDTA in PBS for 45 min followed by gentle polishing withan epithelial scrubber (Amoils Epithelial Scrubber; Innova, InnovativeExcimer Solutions, Inc., Toronto, Ontario, Canada) as previouslydescribed 62. After rinsing with sterile HBSS for 3 times, these AM werestored for 3 to 4 days in DMEM/F12 before seeding with RPE cells.

Passage of RPE Cells

First passage of RPE cells were obtained by the treatment of 0.05%trypsin and 0.02% EDTA in Ca²⁺ and Mg²⁺-free HBSS for 8 min when theywere in the late log phase, and seeded at 5,000-20,000 viable RPE cellsper cm2 (as assessed by trypan blue staining) in DMEM/F12 with Ca²⁺concentrations adjusted to 0.1 mM, and supplemented with 10% dialyzedFBS, 100 IU/ml PNS and 0.5 (μg/ml amphotericin B on either plastic,denuded AM or intact AM. Cells were left undisturbed for 36 h to allowattachment, and culture media were changed biweekly thereafter.

Calcium Switch

When RPE cells on each of the above culture reached confluence, the Ca²⁺concentration in the culture medium was changed to 1.8 mM by addingsoluble calcium salt.

Evaluation

Each culture was followed with observation under a phase contrastmicroscope at 36 h after plating, at confluence, and one week and fourweeks after the calcium switch. At different intervals, cultures wereterminated by removing the medium, and rinsing the cells 3 times withsterile PBS. Thereafter, the cultures were fixed either in pre-cooled(−20° C.) methanol for 5 min for Cytokeratins or in 4% paraformaldehydeat 4° C. for 10 min (for RPE-65, ZO-1). This was followed again byrinsing 3 times in PBS. The tissue was thereafter stored at 4° C. in0.01 NaN3 in PBS for about 2 weeks until further processing. Primaryantibodies were incubated overnight at 4° C. at the followingconcentrations: Pancytokeratin (K8.13) at 1/100, Cytokeratin 18 (CY-90)1/3000 (both according to [25]), ZO-1 at 1/200 and the RPE-65 at 1/200.This was followed by 3 washes in PBS and then incubated for 2 h withrespective secondary antibodies conjugated to either FITC (for RPE-65),Alexa 546 (for ZO-1), Alexa 488 (for Pancytokeratin and Cytokeratin 18)in a dilution of 1/200. All antibodies were diluted in PBS containing 1%BSA and 0.1% Triton x-100. The specimens were washed 3 times in PBS andmounted immediately in Aqua-Poly/mount mounting medium. The staining wasthen analyzed with a Zeiss Axiophot fluorescence microscope (Zeiss,Oberkochen, Germany) which was connected to a CCD Optronics camera. Theimages were subsequently enhanced with Adobe Photoshop 5.5 software. Forimmunostaining, the specificity of the above antibodies had beenverified in dutch belted rabbit tissue. In brief, an animal, previouslyused for another procedure requiring subsequent euthanasia, was perfusedunder anaesthesia with 4% formaldehyde. Thereafter an injection of 4%paraformaldehyde in PBS was given intravitreally and the eye immersed inthe same fixative and kept on ice for 2 h. The anterior segment was thendissected away and the vitreous removed as much as possible. Sampleswere cut with ophthalmic scissors from the posterior pole of the eye andthe cornea, incubated in PBS with sequential sucrose gradients of 10, 20and 30%, embedded in OCT and snap-frozen with liquid nitrogen. Tissuesections were cut at 8 μm on a Reichert Cryostat.

RESULTS

Morphological Appearance of RPE Grown in Low Ca²⁺ on DifferentSubstrates

Rabbit RPE cells were seeded at about 5,000 to about 20,000 viable RPEcells per cm² on plastic (P), epithelially denuded human amnioticmembrane (dAM), or intact human amniotic membrane (iAM in low Ca²⁺DMEM/F12. Confluence was reached in about 7-9 days on dAM, which wasfaster than 9-10 days on plastic. RPE cells on these three cultures ingeneral appeared spindle-shaped and were spread evenly on bothsubstrates except that RPE cells on iAM appeared to be more squamous andpolygonal and less evenly distributed when compared to the plasticculture and dAM when confluence was reached. Furthermore, pigmentationof melanolipofuscin granules rapidly disappeared on plastic cultures inpart due to dilution by cell division, a phenomenon that has beenwell-recognized 33. Nevertheless, RPE cells on dAM still retained somegranular appearance although pigmentation was also reduced, whereas RPEcells on iAM still possessed heavy pigmentation.

Morphological Appearance of RPE One Week After Ca²⁺ Switch

When RPE cells reached confluence on dAM, the medium was switched tohigh Ca²⁺ DMEM/F12. One week after Ca²⁺ switch, RPE cells on plasticcultures remained spindle-shaped, but did not form clear granules exceptsome pigmentation might reappear. In contrast, RPE cells grown on dAMadopted epithelioid appearance with a polygonal (hexagonal) shape, andexhibited abundant granules with pigmentation in the cytoplasm. RPEcells on iAM also adopted polygonal shape with heavy pigmentation. As acomparison, RPE cells grown on dAM and low calcium still retainedspindle shape but appeared to have more pigmentation.

Characterization of Resultant Epithelial Phenotypes by Immunostaining

CK 18 Staining

Staining by antibody to CK 18 (cytokeratin 18), a marker to identify theepithelial origin of RPE cells, was performed. The results indicate thatindeed RPE cultured on plastic under low Ca²⁺ concentration had theepithelial origin because all cells were positive. RPE growing on intactand denuded membrane showed strong positive staining with vividcytoskeleton pattern, that was more pronounced then the counterpartgrowing on plastic when Ca²⁺ was elevated.

RP 65 Staining

Staining to RP65, a new marker for RP differentiation, was performed. Anormal pattern of positive staining of RPE in vivo rabbit retina wasobtained, the photograph of the staining showing a monolayer of RPEbetween the photoreceptors (on the top) and choriocapillaris (on thebottom), and the RPE cells were pigmented.

RP 65 Staining

RP65 staining showed that RPE grown on plastic was negative even 1.5weeks after Ca ²⁺ switch. In contrast, RPE cells were strongly positiveto RP 65 when grown on intact AM and on denuded AM. This resultcontinued when the culture was extended to 3.5 weeks. The positivestaining is shown by green fluorescence, while the reddish staining wasnuclear counterstaining by PI.

ZO-1 Staining

ZO-1 staining is directed to the tight junction complex formed by RPE.In vivo, this antibody to ZO-1 showed positive (reddish fluorescence) inthe RPE and photoreceptor complex. ZO-1 staining was also positive onthe RPE cells grown on plastic and intact amniotic membrane, and ondenuded AM.

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EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents to the specificembodiments described herein. Such equivalents are intended to beencompassed in the scope of the following claims.

1. A method for treating a retinal disease, comprising inserting in asubretinal space of a human patient in need thereof a compositecomprising amniotic membrane and non-immortalized human retinal pigmentepithelial cells or non-immortalized human retinal pigment epithelialequivalent cells on the membrane, wherein the number of non-immortalizedhuman retinal pigment epithelial cells or non-immortalized human retinalpigment epithelial equivalent cells on the membrane is from about 16,000to about 20,000 per 4 mm² of amniotic membrane.
 2. The method of claim1, wherein the retinal disease that is treated is selected from thegroup consisting of retinal detachment, gyrate atrophy, choroideremia,and age-related macular degeneration.
 3. The method of claim 1, whereinthe amniotic membrane is human amniotic membrane.
 4. The method of claim1, wherein the retinal pigment epithelial cells comprise retinal pigmentepithelial cells cultured on the amniotic membrane.
 5. The method ofclaim 1, wherein the composite further comprises a pharmaceuticallyactive molecule.
 6. The method of claim 5, wherein the pharmaceuticallyactive molecule is selected from the group consisting of growth factors,enzymes, and therapeutic drugs.
 7. The method of claim 1, wherein theamniotic membrane is epithelially denuded.
 8. The method of claim 1,wherein the amniotic membrane is intact amniotic membrane comprising abasement membrane and a stroma.
 9. The method of claim 8, whereinmesenchymal cells are added to at least one side of the stroma beforeinsertion in a subretinal space of a patient.
 10. The method of claim 9,wherein the mesenchymal cells are fibroblasts.
 11. The method of claim1, wherein the amniotic membrane is treated on at least one side withexcimer laser ablation before insertion in a subretinal space of apatient.
 12. The method of claim 11, wherein the excimer laser ablationalters the thickness of the stromal side or basement membrane side ofthe amniotic membrane.
 13. The method of claim 1, wherein the retinalpigment epithelial equivalent cells comprise cells selected from thegroup consisting of iris pigment epithelial cells, retinal pigmentepithelial cells differentiated from at least one adult or embryonalstem cell, cells derived from neural retinal cells, and cells derivedfrom a ciliary body.
 14. The method of claim 6, wherein thepharmaceutically active molecule is a growth factor selected form thegroup consisting of retinal pigment epithelium-derived growth factor,transforming growth factor-beta, and interleukin-10.
 15. The method ofclaim 1 wherein the composite is formed by: (a) applying at least oneretinal pigment epithelial cell or retinal pigment epithelial equivalentcell to an amniotic membrane; and (b) culturing the retinal pigmentepithelial cell or retinal pigment epithelial equivalent cell on themembrane under conditions suitable for growth for a period of timesufficient to produce a plurality of cultured cells.